Progress in purifying the opiate receptor, a goal essential to understanding both its chemical nature and function, has been slow. Current evidence suggests that this may be due to the fact that the opiate receptor is a complex, multi-component system, containing differently selective binding sites, regulatory molecules, effector system, closely associated with a proper membrane lipid environment. Solubilization of membrane would be expected to dissociate these components, whose integration may be necessary for full expression of opiate binding as well as biologic activity. We accordingly propose to isolate different receptor components and test the ability of combinations of them to exhibit opiate binding, and, when reconstituted back into the membrane, opiate-mediated function, (e.g., inhibition of adenylate cyclase). The reconstitution step serves as an ultimate test of the integrity of the solubilized receptor and because of its multicomponent nature, may be necessary for full expression of binding activity. In isolation of opiate receptors, our approaches involve detergent solubilization, isolation of binding components by affinity chromatography, and receptor reconstitution. The latter emphasis on both reconstitution of binding and functional activities. Reconstitution methods used include dialysis, sonication and fusion with genetic mutants of S49 lymphoma cells. We will also use a newer approach, solubilizing with the natural detergent lysophosphatidylcholine and reconstituting the membrane by its enzymatic conversion to phosphatidylcholine. This method for the reconstitution of brain membrane, developed in our laboratory, by itself should be very important in the studies of general membrane structure and function. To make our approach thorough, we will also attempt to isolate receptor molecules through the use of monoclonal antibodies, using partially purified receptor to develop the antibodies.